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. 2016 Feb 24;7(14):18171–18182. doi: 10.18632/oncotarget.7685

Figure 2. The expression of downstream molecules in OTS167-treated cells.

Figure 2

A. DEPDC1 expression in MELK knocked-down cells. Oligo siRNA for luciferase (control), MELK or DEPDC1 was transfected into MDA-MB-231 cells. After 24 hours of incubation, protein was detected by western blot analysis. siLuc; si-Luciferase. B. In vivo phosphorylation assay of DEPDC1. COS7 cells were co-transfected with DEPDC1 and either MELK (wt or D150A) or control mock vector. After 24 hours of incubation, cells were treated with okadaic acid for 3 hours. Proteins were separated by Phos-tag PAGE gel to detect phosphorylation of DEPDC1. For lambda phosphatase assay, proteins were incubated with lambda phosphatase before loading onto the gel. wt; wild-type MELK, D150A; kinase-dead mutant MELK, λPP; lambda phophatase. C. The expression of proteins in OTS167-treated cells. MDA-MB-231 cells were incubated with OTS167 for 24 hours at given concentration. MELK, DEPDC1, Slug, E-cadherin and ACTB were detected using specific antibodies. ACTB served as a protein-loading control.